1. Field of the Invention
The present invention relates to the field of genetically engineered peptide production in plants, more specifically, to the use of tobamovirus vectors to express fusion proteins, and more specifically, to a vaccine comprising an encapsidated virus having a modified coat protein which displays a parvovirus antigen.
2. Description of the Background Art
Peptides are a diverse class of molecules having a variety of important chemical and biological properties. Some examples include; hormones, cytokines, immunoregulators, enzyme inhibitors, vaccine antigens, adhesion molecules, receptor binding domains, and the like. The cost of chemical synthesis limits the potential applications of synthetic peptides for many uses such as therapeutic drugs or vaccines. There is a need for inexpensive and rapid synthesis of milligram and larger quantities of naturally occurring polypeptides. Towards this goal many animal and bacterial viruses have been used successfully as peptide carriers.
The safe and inexpensive culture of plants provides an advantageous alternative for cost-effective production of pharmaceutically useful peptides. During the last decade, considerable progress has been made in expressing foreign genes in plants. Foreign proteins arc now routinely produced in many plant species either for modification of the plant or for protein extraction and production. Animal proteins have been effectively produced in plants (reviewed in Krebbers, E. et al., In: Plant Protein Engineering (P. R. Shewry et al., eds.), Cambridge University Press, Cambridge, 1992, pp. 316–324).
Vectors for the genetic manipulation of plants have been derived from several naturally occurring plant viruses, including tobacco mosaic virus (TMV). TMV is the type member of the tobamovirus group. TMV has straight tubular virions of approximately 300×18 nm with a 4 nm-diameter hollow canal, consisting of approximately 2000 units of a single capsid protein wound helically around a single RNA molecule. Virion particles are 95% protein and 5% RNA by weight. The genome of TMV is composed of a single-stranded RNA of 6395 nucleotides containing five large open reading frames (ORFs). Expression of each gene is regulated independently. The virion RNA serves as the messenger RNA (mRNA) for the 5′ genes, encoding the 126 kDa replicase subunit and the overlapping 183 kDa replicase subunit that is produced by read-through of a UAG stop codon approximately 5% of the time. Expression of the internal genes is controlled by different promoters on the minus-sense RNA that direct synthesis of 3′-coterminal subgenomic mRNAs which are produced during replication (FIG. 1). A detailed description of tobamovirus gene expression and life cycle can be found, among other places, in Dawson and Lehto, Adv. Vir. Res. 38:307–342 (1991). It is of interest to provide new and improved vectors for the genetic manipulation of plants.
For production of specific proteins, transient expression of foreign genes in plants using virus-based vectors has several advantages. Products of plant viruses are among the highest produced proteins in plants. Often a viral gene product is the major protein produced in plant cells during virus replication. Many viruses are able to spread quickly from an initial infection site to almost all cells of the plant. For these reasons, plant viruses have been developed into efficient transient expression vectors for foreign genes in plants. Viruses of multi-cellular plants are relatively small, probably due to the size limitation in the pathways that allow viruses to move to adjacent cells in systemic infection of the entire plant. Most plant viruses have single-stranded RNA genomes of less than 10 kb. Genetically altered plant viruses provide one efficient means of transfecting plants with genes encoding peptide-carrier fusion proteins. A discussion of TMV coat protein fusions is provided in Turpen et al., U.S. Pat. No. 5,977,438 entitled “Production of Peptides in Plants as Viral Coat Protein Fusions.” Nov. 2, 1999. See also: Yusibov V. et al., Proc. Natl. Acad. Sci. USA 94:5784–5788 (1997); Modelska, A et al., Proc. Natl. Acad. Sci. USA 95:2481–2485 (1998).
The pathogenesis of parvovirus infection has been most recently reviewed by Parish, C. R., Baillieres Clin. Haematol. 8:57–71, (1995.). Feline parvovirus (FPV) is closely related to canine parvovirus and the respective diseases are similar in pathogenesis. Parvovirus replicates first in the tonsils, and then spreads to its target cells: mitotically active intestinal crypt epithelial cells and bone marrow stem cells. Viremia lasts for less than 7 days before death or recovery. Clinical signs in cats include fever, vomiting, diarrhea, panleukopenia, acute shock and death. The disease outcome is proportional to the severity of the leukopenia; cats with severe panleukopenia will often die, while those with mild leukopenia will usually survive.
The VP2 (or E2) epitope of mink enteritis virus (MEV), which is closely related to FPV, has been previously expressed on the surface of cowpea mosaic virus, which was propagated on the leaves of the black-eyed bean (Dalsgaard, K et al., Nature Biotechnol. 15:248–252 (1997)). One mg of the cow pea mosaic virus material that expressed this epitope was used to immunize minks against virulent MEV. The minks were protected against clinical disease, and shed very little virus. The authors suggested that this epitope, expressed in this manner, could also be used to protect cats and dogs against their respective parvovirus infections.
The coding sequence for VP2 (E2) and the rabies spike glycoprotein have also been engineered into raccoon poxvirus to make a five recombinant vaccine against rabies and feline panleukopenia (Hu, L. et al., 1996. Virology 218:248–252., Hu, L. et al., 1997, Vaccine 15:1466–1472.). Cats vaccinated with this construct showed excellent protection against virulent parvovirus challenge.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.